Tularemia is a fatal human disease caused by highly virulent and category A select agent Francisella tularensis (Ft). No FDA approved vaccine is currently available for tularemia prophylaxis. The history of Ft weaponization and the recent concerns about its use as a bioweapon/bioterror agent has potentiated an urgent need for the development of a safe and effective vaccine. In our previous studies, we have developed a novel approach for the delivery of multiple immunogenic proteins of Ft using Tobacco Mosaic Virus (TMV) as a unique delivery platform. This platform allowed conjugation of a single immunodominant protein antigen directly to the surface of TMV. A cocktail of four protein-TMV conjugates was then blended into a single vaccine formulation and administered intranasally (i.n.). This approach effectively stimulated respiratory immunity, induced potent humoral and cellular immune responses, and provided 100% protection in C57BL/6 mice against i.n. challenge with Ft. We have re-established outbred New Zealand White (NZW) rabbits as a relevant model of human tularemia caused by inhalation of highly virulent Ft SchuS4 strain and have developed a standardized and reproducible method for an aerosol challenge with Ft SchuS4. Tularemia falls under the FDA?s ?Animal Rule? which allows for the demonstration of vaccine efficacy in multiple animal species with an infection process that mimics human disease when human clinical trials are not ethnically or logistically possible. Mice, rats, rabbits, and non-human primates (NHP) all serve as important animal models for tularemia vaccine studies. Mice are often used for initial development and screening of potential vaccine candidates, but the susceptibility of mice to attenuated strains of Ft is a concern in translating these results to NHP or human. NHP studies are logistically and technically challenging, as well as prohibitively expensive. Rat and rabbit models fill the gap in between mouse and NHP models. Outbred NZW rabbits have a pulmonary architecture similar to human and present a clinical picture comparable to that of the human tularemia. Having demonstrated protection in mice, the goal of this proposal is to demonstrate the efficacy of TMV-tularemia subunit vaccine in outbred rabbits against aerosol challenge with the highly virulent Ft SchuS4 strain. We hypothesize that the TMV-tularemia vaccine will elicit strong immunity and protection in the NZW rabbit model. To test this hypothesis, in aim 1, we propose to investigate the protective efficacy of TMV-tularemia vaccine in rabbits and in specific aim 2 we will investigate the immune mechanisms in rabbits vaccinated with fully protective TMV-tularemia vaccine. At the conclusion of these studies, we intend to identify a lead tularemia vaccine formulation that could be developed into a safe, affordable, and potent human vaccine. Our goal is to generate sufficient data in support of TMV tularemia subunit vaccine that will transition our future studies beyond the scope of R21 to undertake clinical trials in NHP models for its further development as an effective human vaccine.